ABSTRACT Elucidation of underlying disease mechanisms in Lewy body dementia requires the systematic characterization of disease-specific changes in LBD brain tissue. The combination of dementia and motor symptoms at the clinical level is reflected in the neuropathology by the accumulation of alpha-synuclein (?-syn) in Lewy bodies and beta-amyloid (A?) in amyloid plaques. Both A? and ?-syn are capable of aggregating into oligomers, fibrils, and beta sheets, but the aggregates that form are extremely heterogeneous in terms of both structure and function. The identification of which specific subspecies of the proteins are neurotoxic or support aggregation is poorly understood and as a result development of diagnostics and treatments that depend on knowledge of protein structure has been slow. Recent studies have suggested a ?seeded? or ?prion-like? propagation for both ?-syn and A?, indicating that abnormal conformations may ?spread? from diseased to healthy cells. While synthetic seeds of ?-syn and A? confer toxicity in cells through various mechanisms, it is well known that the exact method of preparing these species influences their bioactivity to a high degree. Herein we take the next important step to analyze the actual subspecies from post-mortem brains, to define and compare aggregates, and identify mechanisms of toxicity and spreading underlying the selective regional vulnerabilities. We will extract soluble and insoluble ?-syn and A? species from neuropathologically confirmed LBD brains with or without genetic LBD risk factors for structural and functional characterization. We will assess load and distribution of ?-syn and A? subspecies and will detail their characteristics including size, structure and self- templating capabilities (Aim 1). The critical involvement of autophagy-lysosomal pathways in LBD is emphasized by the underlying genetic risks for LBD. Besides mutations in glucocerebrosidase (GBA) that interfere with lysosomal functions, it is well established that individuals with the APOE ?4 allele have a 6-fold greater risk for DLB. We will assess alterations in lysosome function in LBD brain and determine their response and contribution to processing, aggregation, and toxicity of ?-syn and A? subspecies (Aim 2). We will use patients? iPSC-derived neurons with different genetic risk factors to functionally validate the contribution to aggregation, toxicity and spreading of ?-syn and A? (Aim 3). The expertise of this group of investigators synergizes to identify differences in structure and function of subspecies, to investigate mechanisms of aggregation and toxicity, and to unravel the contributions of the genetic variations and alterations in selective autophagy pathways to regional vulnerabilities. This puts us in a unique position to complement the studies of Projects 1, 2, and 3 of this CWOW at multiple levels and towards the full characterization of ?-syn and A? subspecies.